Introduction

Analyse d’un screening CRISPR dans des cellules tryptophane neg. Raphaële a fourni un fichier word détaillé qui explique la manip et les algorithmes utilisés.

Il y a trois fichiers MLE et trois RRA (triplicats biologiques) qui contiennent chacun un b score = une moyenne, un z score = un score qui prend en compte la moyenne et la déviation standard et des pval + wald tests + fdr.

Je vais pool les trois fichiers, avoir le score z des triplicats et déterminer les outliers puis les comparer à ceux que Raphaële a (pour contrôler que je fais pas du caca). Ensuite je prendrai les outliers recroisés entre le MLE et RRA pour pouvoir produire les différentes figures dont Raphaële a besoin.

files = list.files("./data/", pattern = "*mle_trp*")

for (i in 1:length(files)){
  file = read_xlsx(paste0("./data/",files[i]))
  file = file %>% filter(sgRNA >=4) %>% 
    dplyr::select(Gene, `treatment|beta`,`treatment|p-value`)
  colnames(file) = c("Gene", paste0("mean_",i), paste0("pval_",i))
  assign(paste0("mle_",i), file)
}

data = list(mle_1, mle_2, mle_3) %>% 
  purrr::reduce(full_join)

data_all = 
  data %>% 
  drop_na() %>% 
  rowwise() %>% 
  mutate(mean = mean(c(mean_1, mean_2, mean_3)),
         sd = sd(c(mean_1, mean_2, mean_3)),
         pval = mean(c(pval_1, pval_2, pval_3))) %>% 
  dplyr::select(Gene, mean, sd, pval)

# Replacing the pvalues = 0 into pval = 10^-5.5 to fit on the graph

data_all[data_all$pval==0,]$pval = 10^-5.5

data = 
  data_all %>% 
    filter(pval<= 0.05)

boxplot(data$mean)

median(data$mean)

## [1] 0.4738267

outliers = 
  data %>% 
  filter(mean < quantile(data$mean)[2]|mean > quantile(data$mean)[4])

down = 
  data %>% 
  filter(mean < -0.3)

outliers %>% 
  head(15) %>% 
  knitr::kable()

Gene	mean	sd	pval
Mrpl28	1.1918667	0.1163599	0.0000032
Ndufa1	1.1643667	0.0969317	0.0000181
37316	1.0765400	0.1310024	0.0000032
Mrpl20	1.1647333	0.0715976	0.0000032
Ndufa2	1.1362333	0.0476430	0.0000032
Ptplb	0.9026700	0.3572377	0.0004527
Mrps25	1.1595667	0.1321792	0.0000032
Agpat1	1.0188100	0.1424605	0.0000181
Hsd17b10	0.9095200	0.1850416	0.0000362
Ndufc2	1.1130333	0.0349172	0.0000032
Gnb2l1	1.0756667	0.0651772	0.0000032
Mrpl41	0.9315833	0.1756950	0.0000181
Dbr1	0.9692067	0.1070501	0.0000181
Tufm	0.8323300	0.2199795	0.0000543
Ncl	0.6533667	0.3726756	0.0087642

dim(outliers)

## [1] 288   4

Quand je filtre pour pval < 0.05, j’ai 288 outliers pour les MLE compparé aux 261 de Raphaële. On va partir de la liste des outliers MLE+RRA qu’elle a fournie pour la suite.

Possible plots

MLE : pval and mean

RRA : pval and mean

Il faudrait retrouver les fichiers avec les pvaleurs, je peux pas plotter sinon.

Number of gene attributed at random

Number of gene attributed not at random

ORA

Gene ontology

On the 577 significant genes (not specifically the outliers)

ID	Description	GeneRatio	BgRatio
GO:0140053	mitochondrial gene expression	55/538	95/15811
GO:0032543	mitochondrial translation	48/538	70/15811
GO:0010257	NADH dehydrogenase complex assembly	35/538	47/15811
GO:0032981	mitochondrial respiratory chain complex I assembly	35/538	47/15811
GO:0033108	mitochondrial respiratory chain complex assembly	39/538	72/15811
GO:0007005	mitochondrion organization	72/538	456/15811
GO:0006399	tRNA metabolic process	42/538	155/15811
GO:0009060	aerobic respiration	38/538	128/15811
GO:0006119	oxidative phosphorylation	31/538	84/15811
GO:0045333	cellular respiration	42/538	178/15811

On the 177 positive outliers

ONTOLOGY	ID	Description	GeneRatio	BgRatio	pvalue
BP	GO:0010257	NADH dehydrogenase complex assembly	28/170	35/538	0.00e+00
BP	GO:0032981	mitochondrial respiratory chain complex I assembly	28/170	35/538	0.00e+00
BP	GO:0033108	mitochondrial respiratory chain complex assembly	29/170	39/538	0.00e+00
BP	GO:1901566	organonitrogen compound biosynthetic process	78/170	166/538	4.00e-07
BP	GO:0006412	translation	59/170	116/538	8.00e-07
BP	GO:0043043	peptide biosynthetic process	59/170	117/538	1.10e-06
BP	GO:0043604	amide biosynthetic process	60/170	122/538	2.80e-06
BP	GO:0006518	peptide metabolic process	59/170	120/538	3.70e-06
BP	GO:0034645	cellular macromolecule biosynthetic process	62/170	130/538	7.20e-06
BP	GO:0043603	cellular amide metabolic process	61/170	129/538	1.31e-05

On the 144 negative outliers

ONTOLOGY	ID	Description	GeneRatio	BgRatio	pvalue
BP	GO:0006357	regulation of transcription by RNA polymerase II	24/71	57/538	0e+00
BP	GO:0065007	biological regulation	56/71	256/538	0e+00
BP	GO:0006366	transcription by RNA polymerase II	25/71	63/538	0e+00
BP	GO:0006355	regulation of transcription, DNA-templated	27/71	73/538	0e+00
BP	GO:1903506	regulation of nucleic acid-templated transcription	27/71	73/538	0e+00
BP	GO:2001141	regulation of RNA biosynthetic process	27/71	73/538	0e+00
BP	GO:0051252	regulation of RNA metabolic process	30/71	88/538	0e+00
BP	GO:0050789	regulation of biological process	53/71	238/538	0e+00
BP	GO:0019219	regulation of nucleobase-containing compound metabolic process	32/71	103/538	1e-07
BP	GO:0050794	regulation of cellular process	51/71	230/538	1e-07

Reactome

On the 577 significant genes (not specifically the outliers)

ID	Description	GeneRatio	BgRatio	pvalue	p.adjust
R-MMU-5389840	Mitochondrial translation elongation	73/369	83/8497	0.0e+00	0.0000000
R-MMU-5368287	Mitochondrial translation	74/369	86/8497	0.0e+00	0.0000000
R-MMU-5419276	Mitochondrial translation termination	73/369	85/8497	0.0e+00	0.0000000
R-MMU-72766	Translation	88/369	220/8497	0.0e+00	0.0000000
R-MMU-163200	Respiratory electron transport, ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins.	53/369	116/8497	0.0e+00	0.0000000
R-MMU-1428517	The citric acid (TCA) cycle and respiratory electron transport	60/369	163/8497	0.0e+00	0.0000000
R-MMU-6799198	Complex I biogenesis	38/369	55/8497	0.0e+00	0.0000000
R-MMU-611105	Respiratory electron transport	46/369	93/8497	0.0e+00	0.0000000
R-MMU-189451	Heme biosynthesis	7/369	14/8497	7.0e-07	0.0000543
R-MMU-163210	Formation of ATP by chemiosmotic coupling	7/369	17/8497	3.7e-06	0.0002245

On the positive 177 outliers

ID	Description	GeneRatio	BgRatio	pvalue	p.adjust
R-MMU-5419276	Mitochondrial translation termination	53/131	85/8497	0e+00	0.0e+00
R-MMU-5368287	Mitochondrial translation	53/131	86/8497	0e+00	0.0e+00
R-MMU-5389840	Mitochondrial translation elongation	52/131	83/8497	0e+00	0.0e+00
R-MMU-72766	Translation	54/131	220/8497	0e+00	0.0e+00
R-MMU-163200	Respiratory electron transport, ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins.	38/131	116/8497	0e+00	0.0e+00
R-MMU-1428517	The citric acid (TCA) cycle and respiratory electron transport	40/131	163/8497	0e+00	0.0e+00
R-MMU-6799198	Complex I biogenesis	28/131	55/8497	0e+00	0.0e+00
R-MMU-611105	Respiratory electron transport	32/131	93/8497	0e+00	0.0e+00
R-MMU-163210	Formation of ATP by chemiosmotic coupling	6/131	17/8497	1e-07	2.2e-06
R-MMU-8949613	Cristae formation	6/131	17/8497	1e-07	2.2e-06

On the negative 144 outliers

ID	Description	GeneRatio	BgRatio	pvalue	p.adjust
R-MMU-392518	Signal amplification	4/35	32/8497	0.0000080	0.0023261
R-MMU-428930	Thromboxane signalling through TP receptor	3/35	23/8497	0.0001072	0.0118473
R-MMU-418592	ADP signalling through P2Y purinoceptor 1	3/35	24/8497	0.0001221	0.0118473
R-MMU-418597	G alpha (z) signalling events	3/35	31/8497	0.0002659	0.0154777
R-MMU-456926	Thrombin signalling through proteinase activated receptors (PARs)	3/35	31/8497	0.0002659	0.0154777
R-MMU-8939211	ESR-mediated signaling	5/35	158/8497	0.0004315	0.0207295
R-MMU-977444	GABA B receptor activation	3/35	40/8497	0.0005699	0.0207295
R-MMU-991365	Activation of GABAB receptors	3/35	40/8497	0.0005699	0.0207295
R-MMU-3371568	Attenuation phase	2/35	13/8497	0.0012496	0.0389705
R-MMU-9006931	Signaling by Nuclear Receptors	5/35	208/8497	0.0014950	0.0389705

Highlight genes per found role

Outliers found in GO terms (BP and CC)

ONTOLOGY	ID	Description	GeneRatio	BgRatio	pvalue
BP	GO:0010257	NADH dehydrogenase complex assembly	28/170	35/538	0.0000000
BP	GO:0032981	mitochondrial respiratory chain complex I assembly	28/170	35/538	0.0000000
BP	GO:0033108	mitochondrial respiratory chain complex assembly	29/170	39/538	0.0000000
BP	GO:1901566	organonitrogen compound biosynthetic process	78/170	166/538	0.0000004
BP	GO:0006412	translation	59/170	116/538	0.0000008
BP	GO:0043043	peptide biosynthetic process	59/170	117/538	0.0000011
BP	GO:0043604	amide biosynthetic process	60/170	122/538	0.0000028
BP	GO:0006518	peptide metabolic process	59/170	120/538	0.0000037
BP	GO:0034645	cellular macromolecule biosynthetic process	62/170	130/538	0.0000072
BP	GO:0043603	cellular amide metabolic process	61/170	129/538	0.0000131
BP	GO:0006119	oxidative phosphorylation	21/170	31/538	0.0000223
BP	GO:0007005	mitochondrion organization	38/170	72/538	0.0000487
BP	GO:0140053	mitochondrial gene expression	31/170	55/538	0.0000525
BP	GO:0032543	mitochondrial translation	28/170	48/538	0.0000551
BP	GO:0043933	protein-containing complex organization	48/170	99/538	0.0000752
BP	GO:0045333	cellular respiration	25/170	42/538	0.0000932
BP	GO:0009060	aerobic respiration	23/170	38/538	0.0001293
BP	GO:0015980	energy derivation by oxidation of organic compounds	25/170	43/538	0.0001609
BP	GO:0065003	protein-containing complex assembly	45/170	95/538	0.0002875
BP	GO:0022900	electron transport chain	17/170	27/538	0.0005729
BP	GO:0022904	respiratory electron transport chain	16/170	25/538	0.0006507
BP	GO:0019646	aerobic electron transport chain	13/170	19/538	0.0008742
BP	GO:0022607	cellular component assembly	52/170	120/538	0.0014491
BP	GO:0046034	ATP metabolic process	21/170	38/538	0.0014779
BP	GO:0042773	ATP synthesis coupled electron transport	14/170	22/538	0.0016035
BP	GO:0042775	mitochondrial ATP synthesis coupled electron transport	14/170	22/538	0.0016035
CC	GO:0005739	mitochondrion	138/171	253/541	0.0000000
CC	GO:0098798	mitochondrial protein-containing complex	90/171	128/541	0.0000000
CC	GO:0000313	organellar ribosome	53/171	72/541	0.0000000
CC	GO:0005761	mitochondrial ribosome	53/171	72/541	0.0000000

Organize by theme

BP Mitochondrial gene expression (encompasses mitochondrial translation)
BP ATP metabolic process (encompasses proton-motive force driven ATP prod)
CC respiratory chain complex I
BP oxidative phosphorylation

Outliers found in reactome

ID	Description	GeneRatio	BgRatio
R-MMU-5419276	Mitochondrial translation termination	53/132	85/8497
R-MMU-5368287	Mitochondrial translation	53/132	86/8497
R-MMU-5389840	Mitochondrial translation elongation	52/132	83/8497
R-MMU-72766	Translation	54/132	220/8497
R-MMU-163200	Respiratory electron transport, ATP synthesis by chemiosmotic coupling, and heat production by uncoupling proteins.	39/132	116/8497
R-MMU-1428517	The citric acid (TCA) cycle and respiratory electron transport	41/132	163/8497
R-MMU-6799198	Complex I biogenesis	29/132	55/8497
R-MMU-611105	Respiratory electron transport	33/132	93/8497
R-MMU-163210	Formation of ATP by chemiosmotic coupling	6/132	17/8497
R-MMU-8949613	Cristae formation	6/132	17/8497
R-MMU-189451	Heme biosynthesis	5/132	14/8497
R-MMU-1592230	Mitochondrial biogenesis	6/132	27/8497
R-MMU-189445	Metabolism of porphyrins	5/132	31/8497
R-MMU-3371378	Regulation by c-FLIP	2/132	10/8497
R-MMU-5218900	CASP8 activity is inhibited	2/132	10/8497
R-MMU-69416	Dimerization of procaspase-8	2/132	10/8497
R-MMU-1362409	Mitochondrial iron-sulfur cluster biogenesis	2/132	11/8497
R-MMU-202427	Phosphorylation of CD3 and TCR zeta chains	2/132	12/8497
R-MMU-199220	Vitamin B5 (pantothenate) metabolism	2/132	13/8497
R-MMU-389948	PD-1 signaling	2/132	13/8497
R-MMU-140534	Caspase activation via Death Receptors in the presence of ligand	2/132	15/8497
R-MMU-202433	Generation of second messenger molecules	2/132	19/8497
R-MMU-5357769	Caspase activation via extrinsic apoptotic signalling pathway	2/132	19/8497
R-MMU-71403	Citric acid cycle (TCA cycle)	2/132	21/8497
R-MMU-5213460	RIPK1-mediated regulated necrosis	2/132	28/8497
R-MMU-5675482	Regulation of necroptotic cell death	2/132	28/8497
R-MMU-1852241	Organelle biogenesis and maintenance	6/132	212/8497
R-MMU-9706019	RHOBTB3 ATPase cycle	1/132	10/8497
R-MMU-264870	Caspase-mediated cleavage of cytoskeletal proteins	1/132	11/8497
R-MMU-71406	Pyruvate metabolism and Citric Acid (TCA) cycle	2/132	47/8497

Organize and highlight targets by theme and functions

## R version 4.2.2 (2022-10-31 ucrt)
## Platform: x86_64-w64-mingw32/x64 (64-bit)
## Running under: Windows 10 x64 (build 19045)
## 
## Matrix products: default
## 
## locale:
## [1] LC_COLLATE=English_Belgium.utf8  LC_CTYPE=English_Belgium.utf8   
## [3] LC_MONETARY=English_Belgium.utf8 LC_NUMERIC=C                    
## [5] LC_TIME=English_Belgium.utf8    
## 
## attached base packages:
## [1] stats4    stats     graphics  grDevices utils     datasets  methods  
## [8] base     
## 
## other attached packages:
##  [1] org.Mm.eg.db_3.16.0   AnnotationDbi_1.60.0  IRanges_2.32.0       
##  [4] S4Vectors_0.36.0      Biobase_2.58.0        BiocGenerics_0.44.0  
##  [7] ReactomePA_1.42.0     clusterProfiler_4.6.0 msigdbr_7.5.1        
## [10] readxl_1.4.1          ggrepel_0.9.2         forcats_0.5.2        
## [13] stringr_1.4.1         dplyr_1.0.10          purrr_0.3.5          
## [16] readr_2.1.3           tidyr_1.2.1           tibble_3.1.8         
## [19] ggplot2_3.4.0         tidyverse_1.3.2      
## 
## loaded via a namespace (and not attached):
##   [1] shadowtext_0.1.2       backports_1.4.1        fastmatch_1.1-3       
##   [4] systemfonts_1.0.4      plyr_1.8.8             igraph_1.3.5          
##   [7] lazyeval_0.2.2         splines_4.2.2          BiocParallel_1.32.4   
##  [10] GenomeInfoDb_1.34.4    digest_0.6.30          yulab.utils_0.0.6     
##  [13] htmltools_0.5.3        GOSemSim_2.24.0        viridis_0.6.2         
##  [16] GO.db_3.16.0           fansi_1.0.3            magrittr_2.0.3        
##  [19] memoise_2.0.1          googlesheets4_1.0.1    tzdb_0.3.0            
##  [22] Biostrings_2.66.0      graphlayouts_0.8.4     modelr_0.1.10         
##  [25] svglite_2.1.1          timechange_0.1.1       enrichplot_1.18.3     
##  [28] colorspace_2.0-3       rappdirs_0.3.3         blob_1.2.3            
##  [31] rvest_1.0.3            textshaping_0.3.6      haven_2.5.2           
##  [34] xfun_0.34              crayon_1.5.2           RCurl_1.98-1.9        
##  [37] jsonlite_1.8.3         graph_1.76.0           scatterpie_0.1.8      
##  [40] ape_5.6-2              glue_1.6.2             polyclip_1.10-4       
##  [43] gtable_0.3.1           gargle_1.2.1           zlibbioc_1.44.0       
##  [46] XVector_0.38.0         graphite_1.44.0        scales_1.2.1          
##  [49] DOSE_3.24.2            DBI_1.1.3              Rcpp_1.0.9            
##  [52] viridisLite_0.4.1      gridGraphics_0.5-1     tidytree_0.4.2        
##  [55] reactome.db_1.82.0     bit_4.0.4              httr_1.4.4            
##  [58] fgsea_1.24.0           RColorBrewer_1.1-3     ellipsis_0.3.2        
##  [61] pkgconfig_2.0.3        farver_2.1.1           sass_0.4.2            
##  [64] dbplyr_2.2.1           utf8_1.2.2             labeling_0.4.2        
##  [67] ggplotify_0.1.0        tidyselect_1.2.0       rlang_1.0.6           
##  [70] reshape2_1.4.4         munsell_0.5.0          cellranger_1.1.0      
##  [73] tools_4.2.2            cachem_1.0.6           downloader_0.4        
##  [76] cli_3.4.1              generics_0.1.3         RSQLite_2.2.19        
##  [79] gson_0.0.9             broom_1.0.1            evaluate_0.18         
##  [82] fastmap_1.1.0          ragg_1.2.5             yaml_2.3.6            
##  [85] ggtree_3.6.2           babelgene_22.9         knitr_1.41            
##  [88] bit64_4.0.5            fs_1.5.2               tidygraph_1.2.2       
##  [91] KEGGREST_1.38.0        ggraph_2.1.0           nlme_3.1-160          
##  [94] aplot_0.1.9            xml2_1.3.3             compiler_4.2.2        
##  [97] rstudioapi_0.14        png_0.1-7              reprex_2.0.2          
## [100] treeio_1.22.0          tweenr_2.0.2           bslib_0.4.1           
## [103] stringi_1.7.8          highr_0.9              lattice_0.20-45       
## [106] Matrix_1.5-1           vctrs_0.5.0            pillar_1.8.1          
## [109] lifecycle_1.0.3        jquerylib_0.1.4        data.table_1.14.4     
## [112] cowplot_1.1.1          bitops_1.0-7           patchwork_1.1.2       
## [115] qvalue_2.30.0          R6_2.5.1               gridExtra_2.3         
## [118] codetools_0.2-18       MASS_7.3-58.1          assertthat_0.2.1      
## [121] withr_2.5.0            GenomeInfoDbData_1.2.9 parallel_4.2.2        
## [124] hms_1.1.2              grid_4.2.2             ggfun_0.0.9           
## [127] HDO.db_0.99.1          rmarkdown_2.18         googledrive_2.0.0     
## [130] ggforce_0.4.1          lubridate_1.9.0

RAB117_report

Marion Hardy

2023-05-12

Introduction

Possible plots

MLE : pval and mean

RRA : pval and mean

Number of gene attributed at random

Number of gene attributed not at random

ORA

Gene ontology

On the 577 significant genes (not specifically the outliers)

On the 177 positive outliers

On the 144 negative outliers

Reactome

On the 577 significant genes (not specifically the outliers)

On the positive 177 outliers

On the negative 144 outliers

Highlight genes per found role

Outliers found in GO terms (BP and CC)

Outliers found in reactome

Organize and highlight targets by theme and functions